Hi,
as some of you will have seen from the last couple of posts, I am setting up biolistic transformation in my lab. Never one to go strictly according to the book, I am considering using a pha-1 mutant rescue as the basis for my transformation. I have a few questions regarding this idea.
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Although I have found references to using this approach, they are few and far between and I get the feeling also from posts to this forum that it is not a method of choice? Are there specific reasons that I have overlooked…is it a question of ease of phenotype scoring e.g. unc-119 rescue vs. pha-1 rescue, a question of the vectors available or just that other methods have taken precedence since the pha-1 approach became available?
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In terms of transforming the pha-1 strain, I could go the way of bombarding two separate plasmids (rescue + promoter-gene of interest) or one combined (everything in pC1 or pBX), together (or in subsequent experiments) with single-stranded oligos to promote integration.
Are the issues relating to loss of one non-integrated plasmid but not the other or the difficulty in integrating two different plasmids in the same animal the main ones to consider here?
- I had thought about using gfp to mark the transformed animals by creating a translational reporter. One consideration however, is that although I would include the necessary 5’ and 3’ sequences (e.g. in the genomic fragment), joining these sequences (which will mainly be coding for receptors) to gfp might create a falsely targeted or inherently unstable protein.
Does anyone have any basic tips for minimisíng the chances that the gfp tag influences placement of the receptor protein. Of course, I can check for the presence of the construct using PCR, but not where it is expressed…
Regards
Steve